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Neuromuscular Disorders 29 (2019) 704–715 www.elsevier.com/locate/nmd Workshop report

240th ENMC workshop: The involvement of skeletal muscle stem cells in the pathology of muscular dystrophies 25–27 January 2019, Hoofddorp, The Netherlands

a ,b , ∗ c a, b d , ∗ Jennifer Morgan , Gillian Butler-Browne , Francesco Muntoni , Ketan Patel , on behalf of # the skeletal muscle stem cells involvement in pathology study group a University College London Great Ormond Street Institute of Child Health, 30 Guilford Street, London WC1N 1EH, UK b NIHR Great Ormond Street Hospital Biomedical Research Centre, 30 Guilford Street, London WC1N 1EH, UK c Center for Research in Myology, Association Institut de Myologie, Inserm, Sorbonne Université, 75013 Paris, France d School of Biological Sciences, University of Reading, Reading, RG6 6AS, UK Received 28 June 2019

1. Introduction be expressed either only within the muscle fibre to produce components of the dystrophin-associated protein complex, or Twenty-six participants representing patients, funding within cells lying outside the myofibre (e.g., by fibroblasts agencies and basic and clinical scientists involved in research (reviewed [1] ). Skeletal muscle needs to grow, maintain into skeletal muscle stem cells and muscular dystrophies from itself for a lifetime and also repair itself in response to France, Germany, Italy, India, UK, Australia, Spain, USA, injuries or increased load. In muscular dystrophies, skeletal The Netherlands and Switzerland met in Hoofddorp, The muscles undergo chronic intrinsic necrosis that gives rise to Netherlands on 25–27 January 2019. The meeting was held myogenesis and regeneration, in order to replace the necrotic under the auspices of the ENMC and ENMC main sponsors, portion of the myofibres. The satellite cell is the stem cell that with the additional support of Muscular Dystrophy UK. is involved in regeneration as well as skeletal muscle growth The central aim of this workshop was to determine and postnatal myogenesis and possibly hypertrophy. It sits in whether there could be some intrinsic problems with satellite a niche between the myofibre sarcolemma and basal lamina cell function (e.g., activation and myoblast proliferation, both of which regulate satellite cell quiescence, activation and differentiation and fusion) and consequently provide evidence self-renewal. for satellite cell involvement in pathology in major classes Expression of several genes responsible for muscular of muscular dystrophy and congenital myopathies. This is dystrophies and also for congenital myopathies were not most likely to manifest as impaired muscle formation during initially detected in satellite cells. These findings have major development in the congenital myopathies. However, some implications in that they support the idea that muscular problems in muscle formation may be due to extrinsic factors dystrophies do not arise from the malfunctioning of satellite in the microenvironment of the satellite cells/myoblasts, such cells and that correcting other compartments should be as altered composition of the extracellular matrix. sufficient to alleviate the disease. However with the advent Although they vary in many features including severity, of powerful detection methods it is becoming clear that muscle groups affected, age of onset and heart involvement, this conclusion is not generally sustainable. This is best muscular dystrophies are characterised by progressive skeletal exemplified by discoveries made in the field of Duchenne muscle weakness and wasting. They are generally caused Muscular Dystrophy (DMD). The involvement of the satellite by mutations in genes that have been historically thought to cell in DMD was thought to be a secondary response to the lack of dystrophin in the myofibre as well as an excessive proliferation of the satellite cells which in human have ∗ Corresponding authors. limited replicative potential due to telomere shortening. The E-mail addresses: [email protected] (J. Morgan), [email protected] (K. Patel). dystrophin-deficient myofibre undergoes necrosis and satellite # Listed at the end of this report. cells become activated, proliferate and then contribute to https://doi.org/10.1016/j.nmd.2019.07.003 0960-8966 J. Morgan, G. Butler-Browne and F. Muntoni et al. / Neuromuscular Disorders 29 (2019) 704–715 705 muscle regeneration and reconstitution of the satellite cell DMD, this talk first summarised the many persisting problems pool. In DMD, it was long held that the satellite cells with clinical applications of this muscle stem cell therapy fail in their myogenic capacity and ability to proliferate approach that need to be overcome. Key issues include after repeated episodes of muscle necrosis and regeneration. the source of ‘normal donor’ skeletal muscle stem cells, Muscle fibres become progressively replaced by fibrosis, an expansion in tissue culture, best route of delivery, massive adverse environment that also can impair myogenesis. initial death of injected cultured myogenic cells, and limited Recent findings show that dystrophin is expressed proliferation and migration in vivo , before fusion with the transiently in satellite cells and this is proposed to control host dystrophic muscle cells. These major problems need to their asymmetric divisions, so that satellite cells from the be realistically addressed when proposing stem cell therapies dystrophin-deficient mdx mouse model of DMD give rise for muscular dystrophies. Another therapeutic strategy for to a disproportionate number of stem-like satellite cells at delivering myogenic stem cells, is the exciting field of tissue the expense of differentiation-competent myoblast progenitors engineering that aims to build new muscle constructs to [2] . This observation raised the possibility of DMD being a replace muscles that are unable to regenerate due to some muscle stem cell disease. But this may be too simplistic, as impaired intrinsic capacity of muscle stem cells; while this there appears to be excellent myogenesis and new muscle holds great promise for many creative tissue culture studies formation by dystrophic satellite cells during growth of of muscle cells, there remain serious challenges for clinical mdx mice, and in tissue culture, that is maintained in the applications [8] . mdx mouse, even after persistent intrinsic myonecrosis of The critical issue of identifying whether there is any limb muscles over many months and after experimental intrinsic problem with satellite cell capacity for myogenesis muscle injury [3] , suggesting that dystrophin is not critical in DMD specifically, and other muscular dystrophies was then for satellite cell function under the experimental conditions addressed. This can be identified by some marked problems evaluated. with muscle formation during development (congenital The satellite cell dysfunction in mouse models of manifestation): not evident in mdx mice, nor conspicuous dystroglycanopathy [4] and collagen VI deficiency [5] may be for DMD baby boys. Then it is essential to determine due to defects in the environment (niche), rather than intrinsic histologically the extent of any ongoing intrinsic myofibre defects in the satellite cells themselves. But the extent to necrosis (thus the need for regeneration) in growing and adult which members of the DAPC are expressed in satellite cells muscles; however, care is needed when assessing biopsies and whether their expression within satellite cells is vital for since many morphological changes in muscles that can occur satellite cell function, is not clear. over time for other reasons (e.g., split myofibres, central Satellite cells also seem to have some direct effects on myonuclei) can sometimes be misinterpretated as resulting their environment and any disturbance in this may lead to from necrosis/regeneration [9] . If there is extensive intrinsic muscle pathology: it is proposed that satellite cells signal to necrosis, as evident in dystrophic rodent and dog models of fibroblasts to hold them in quiescence and loss of satellite DMD, then the capacity for sustained new muscle formation cells may increase muscle fibrosis. and regeneration needs to be determined: yet it is widely Mutations in genes that regulate satellite cell/myoblast reported that limb muscles of mdx mice have excellent activity are implicated in some congenital muscular sustained new muscle formation. However, one has to be dystrophies [6,7] . Here, altered behaviour of myoblasts prior cautious about concluding that the same properties exist to them fusing to form myofibres during embryogenesis and in the human without further evidence. The capacity for early growth results in impaired muscle formation evident at myogenesis (proliferation and fusion) of the satellite cells birth. can also be examined experimentally; in vitro studies show It is essential that we accurately assess the contribution of excellent myogenesis (equivalent to normal muscles) even for satellite cells to pathology of diverse muscular dystrophies, satellite cells cultured from old mdx mice aged 15 months [3] , since current therapies mostly aim at correcting abnormalities and in vivo studies using experimental muscle injury in mdx solely in the myofibre. This, if not addressed, will have major mice show very similar regenerative capacity for old mdx and implications on the long term efficacy of any treatment; if a normal muscles. defect in satellite cells is either the main, or a contributory, Thus overall, in animal models and possibly in DMD, these cause of a disease, then treating the myofibre will at best be many observations strongly indicate no significant intrinsic partially successful. problem with myogenesis of dystrophic skeletal muscle stem cells in vivo . This clearly contrasts with recent claims [2] that 2. Defects in muscle regeneration in different need to be carefully re-considered in the context of this neuromuscular conditions large body of evidence for excellent intrinsic regeneration of dystrophic muscles. Instead, the central problem for 2.1. Duchenne Muscular Dystrophy: Grounds DMD may be the repeated necrosis of dystrophic muscles over many years, exacerbated by growth for almost 2 Since skeletal muscle satellite (stem) cell, also called decades. While necrosis initially results in good regeneration myoblast, transfer therapy has been intensively investigated of dystrophic muscles, over time the persistent bouts of for over 30 years as a possible gene replacement strategy for asynchronous damage and inflammation progressively change 706 J. Morgan, G. Butler-Browne and F. Muntoni et al. / Neuromuscular Disorders 29 (2019) 704–715 the extracellular matrix composition with increasing collagen 158901) are linked to the hypomethylation of D4Z4 repeats deposition and fibrosis that can impair myogenesis and stop on chromosome 4q35. Each D4Z4 repeat contains an open regeneration, leading to severe loss of skeletal muscle mass reading frame for a retro-transposed transcription factor, and function in DMD boys. double homeobox chromosome 4 (DUX4). Such epigenetic de-repression in FSHD leads to expression of DUX4 from the 2.2. Satellite cell behaviour in myotonic dystrophy: Furling most distal D4Z4 unit, with DUX4 message then stabilized by a polyA signal located in the flanking pLAM region [11] . Myotonic dystrophy type 1 (DM1 or Steinert disease), DUX4 is a homeodomain-containing transcription factor, and one of the most common neuromuscular diseases in adults, its aberrant expression is considered the primary cause of is caused by an unstable expansion of CTG repeats FSHD pathology [11 , 12] .  within the 3 UTR of the DMPK gene. DM1 is an Investigation into how DUX4 drives FSHD pathology RNA dominant disorder in which nuclear-retained DMPK has mainly focused on expression of DUX4 target genes. transcripts containing CUG expansions alter the function However, the homoedomains of DUX4 have high homology of RNA binding proteins. Abnormal binding of MBNL to those of PAX7 and are interchangeable, suggesting an proteins to pathologic CUG tract leads to their sequestration alternate hypothesis whereby ectopic DUX4 expression in within nuclear CUGexp-RNA foci and subsequently, to their FSHD interferes with PAX7 function. DUX4 and PAX7 functional loss that is associated with RNA metabolism mutually inhibit each other in activating their respective alterations including alternative splicing misregulations. transcriptional target genes in human cells [13] , while, PAX7 Interestingly, DM1 splicing changes in CLCN1, BIN1 and can rescue DUX4-mediated apoptosis in murine myoblasts. DMD pre-mRNAs are associated with symptoms such Banerji and Zammit have recently found that the repression as myotonia, muscle weakness and dystrophic process, of PAX7 transcriptional target genes is a hallmark of FSHD, respectively. At the clinical level, five forms of the disease proposing a model whereby DUX4 causes pathology not (late, adult, juvenile, infantile and congenital) are recognized only by directly activating its own target genes, but also based on the occurrence and onset of symptoms as well by interfering with the ability of PAX7 to regulate its as the size of the CTG tract, which correlates with transcriptional gene target cohort [13] . Indeed, PAX7 target severity of symptoms. In addition to myotonia, DM1 skeletal gene repression in FSHD correlates with disease severity, muscles displayed progressive weakness and atrophy as independently of DUX4 target gene expression. At the well as histological features including increased variation of single cell level, PAX7 target gene repression can efficiently fibre diameter, central nuclei, sarcoplasmic masses, nuclear discriminate FSHD cells, even when no DUX4 target genes clumps and ring fibres. Although an increased number of are detectable [14] . Thus, PAX7 target gene repression is a satellite cells are found in the more affected muscles, major biomarker for FSHD. Since DUX4 can be expressed in several studies support altered behaviour of DM1 satellite primary FSHD patient-derived proliferating myoblasts, such cells that could impact their regenerative capacity and/or interference of PAX7 function by DUX4 would affect satellite muscle mass maintenance. Thus, DM1 muscle precursor cells cell mediated repair in FSHD. have a reduced proliferative capacity in vitro due to an Zammit and colleagues also explored FSHD early activation of the p16-dominant pathway leading to pathomechanisms, particularly the pathways that control premature replicative senescence [10] . Moreover, expression mitochondria: of interest considering alterations in of mutant RNAs harbouring large CUG expansions alters mitochondrial structure and function in FSHD muscle, the myogenic differentiation process of DM1 myoblasts. and the known sensitivity of FSHD cells to oxidative Defective differentiation/fusion was observed in myoblasts stress. Notably, they identified suppression of mitochondrial isolated from muscles of severe congenital DM1 patients biogenesis in FSHD, in particular via PGC1alpha, the showing muscle immaturity or delayed muscle maturation. co-factor and activator of ERRalpha. Myoblasts from Altogether these results suggest that satellite cell functions FSHD patients either form hypotrophic myotubes, with are progressively impaired by unstable CTG expansions, a thin, elongated morphology, or myotubes with an a mechanism that could contribute to the gradual muscle unusual distribution of myonuclei and dysregulation of atrophy in DM1 pathology. Additional studies are required the microtubule network. Known ERRalpha agonists and safe to determine the role of extrinsic and/or intrinsic signals on food supplements Biochanin A, Genistein or Daidzein can impaired behaviour of satellite cells in DM1. rescue this hypotrophic myogenic differentiation in FSHD. Thus suppression of the PGC1alpha-ERRalpha axis leads to 2.3. Satellite cells, DUX4, PAX7 and facioscapulohumeral perturbed myogenic differentiation, which can effectively be muscular dystrophy: Zammit rescued by readily-available food supplements, which may lead to a regenerative therapy approach for FSHD. Facioscapulohumeral muscular dystrophy (FSHD) is characterized by a descending skeletal muscle weakness 2.4. Oculopharyngeal muscular dystrophy: Butler-Browne and wasting. This implies that the normal repair functions performed by muscle satellite cells are compromised in The regenerative capacity of skeletal muscle declines with FSHD. Both FSHD1 (MIM 158900) and FSHD2 (MIM age –this decline has been partly attributed to extrinsic J. Morgan, G. Butler-Browne and F. Muntoni et al. / Neuromuscular Disorders 29 (2019) 704–715 707 environmental influences and diverse intrinsic potential of consequence is a disruption of the progressive alpha- the muscle stem cells themselves. Age-related muscle decline dystroglycan glycosylation that physiologically occurs during can be exacerbated in patients with late onset degenerative myogenesis [17] , potentially participating in the myopathic disorders. A progressive loss of function of pharyngeal process. So, the critical role of this O -glycosyltransferase 1 muscles is frequently encountered in ageing subjects (a protein, POGLUT1, in the maintenance of a correct pool condition called achalasia) as well as in several neurological of satellite cells has been uncovered, reinforcing the Notch or neuromuscular disorders. Among them, oculopharyngeal signalling pathway as a potential therapeutic target for muscular dystrophy (OPMD) is a late-onset autosomal this myopathy. Primary myoblast culture and Drosophila dominant degenerative muscle disorder, characterized by have been demonstrated to be useful experimental models weakness of eyelids and pharyngeal muscles that typically to study the effects of POGLUT1 mutations on satellite appear from the fifth decade, leading respectively to ptosis cells. Generation of mouse models are in progress, showing and dysphagia. The genetic mutation - a short polyalanine promising preliminary results. expansion in the ubiquitous PABPN1 protein [15] –was found over 20 years ago but the pathophysiological mechanisms 2.6. Congenital myopathies: Jungbluth leading to the specific alterations of the myogenic program in these muscles remains to be determined. Butler-Browne Jungbluth reviewed the evidence for satellite cell reported that, although sharing similar functions, all muscles involvement in the congenital myopathies (CMs), a are not equal in terms of structure and presence of satellite heterogeneous group of early-onset neuromuscular disorders cells, and among them pharyngeal and eyelid muscles - per conventionally defined by the abundance of certain structural se show atypical features, such as hypotrophic muscle fibres abnormalities – cores, nemaline rods and central nuclei – surrounded by connective tissue that are usually considered on muscle biopsy. In contrast to the congenital muscular to be pathological in the other skeletal muscles (limb dystrophies (CMDs), the plasma membrane integrity is muscles). Surprisingly, during ageing Butler-Browne recently typically preserved, reflected in normal or only mildly demonstrated that pharyngeal muscles have an increased elevated CK levels. More than 20 genes have been implicated proportion of muscle stem cells –6% compared to the 1% or to date, encoding proteins most commonly involved in less classically observed in the limb muscles of ‘old’ subjects. various aspects of excitation-contraction coupling, the process In addition, they observed the presence of many satellite whereby a neuronal impulse is translated into intracellular cells in the interstitial space. In the pharyngeal muscles of calcium release and muscle contraction. Mutations in the OPMD patients the number of muscle stem cells is even skeletal muscle ryanodine receptor (RYR1) gene encoding the higher (12%) compared to control subjects of the same age, principal sarcoplasmic reticulum calcium release channel are whereas there is no signs of ongoing regeneration. Different the most common cause and have been associated with a hypotheses could explain this increased percentage of muscle wide phenotypical spectrum, ranging from severe CMs with stem cells both inside and outside of their niche, including antenatal onset to induced episodic manifestations – malignant extrinsic and/or intrinsic signals. Butler-Browne presented the hyperthermia and (exertional) rhabdomyolysis –in otherwise different techniques used on human muscle samples: gene healthy individuals. expression profile studies, co-culture experiments, xenograft The evidence for satellite cell involvement is strongest in models and CyToF to decipher the potential mechanisms Early-onset Myopathy with Areflexia, Respiratory Distress involved. Preliminary results demonstrate both instrinsic and and Dysphagia (EMARDD) due to recessive mutations in extrinsic signals are involved. The study of the specific MEGF10 , encoding a protein highly expressed in nerve and signature of pharyngeal muscle stem cells which Butler- skeletal muscle tissues, (in particular in activated satellite Browne has observed will further our understanding of what cells) [7 , 18] . The MEGF10 protein plays a role in satellite cell makes this muscle more susceptible to diseases and ageing proliferation, differentiation and fusion into multinucleated and why the large number of muscle stem cell present in these fibres, as supported by the typical muscle biopsy appearance muscles do not help muscle regeneration. Disorders affecting of small and poorly fused myofibers in MEGF10-deficient these muscles will represent particularly informative models states. MEGF10 is also involved in satellite cell self to better understand skeletal muscle stem cell homeostasis in renewal and maintenance of the satellite cell quiescent adults and in ageing. state. An important role in muscle regeneration is evidenced by the dystrophic phenotype in the MEGF10 KO mouse, 2.5. POGLUT1 mutation: Paradas and worsening of dystrophic features in the MEGF10/mdx double KO when compared to the respective single mutants. Padaras discussed recessive mutations in the POGLUT1 Exaggerated satellite cell loss with aging and repeated gene that have been associated with a muscular dystrophy due injury, probably due to a shift to myogenic commitment to decreased Notch signalling and reduced pool of quiescent and a consequently inadequately maintained muscle satellite satellite cells, demonstrated in adult skeletal muscle from cell pool, has also been observed in recessive SEPN1- patients [16] . A direct consequence is a slow proliferation and related myopathies, a group of CMs characterized clinically differentiation shown by the mutant human myoblasts, which by predominant axial involvement with early scoliosis and causes an alteration of the muscle repair process. A secondary respiratory impairment, and increases in connective tissue, 708 J. Morgan, G. Butler-Browne and F. Muntoni et al. / Neuromuscular Disorders 29 (2019) 704–715

fibre type disproportion and cores (“Multi-minicore Disease, to exacerbate disease pathology by backcrossing mdx mice on MmD”) on muscle biopsy. SEPN1 encodes Selenoprotein another genetic background. Based on findings in the gamma- N, a member of a protein group mediating the effects of sacroglycan model for LGMD where pathology became worse Selenium, whose biological functions are not fully understood when crossed on a DBA2/J genetic background [21 , 22] , D2- but appear to include an important role in redox regulation. mdx mice were generated. She discussed the ‘On Mouse And Reflective of the reported satellite cell abnormalities, SelN Measures’ initiative of Charley’s Fund where all available deficiency has been associated with loss of regenerative data of this novel DMD model were collected. In short, capacity, muscle mass and strength on the organismal level pathology is characterized by early onset of degeneration [19] . A reduction in absolute satellite cell numbers has also and regeneration. Overall muscle pathology (necrosis, fibrosis, been reported in muscle biopsies from affected humans and inflammation) is more apparent in the D2- mdx than in animal models of X-linked myotubular myopathy (XLMTM), the BL10- mdx model due to alterations in Ltbp4 (causing specific qualitative studies have not yet been performed [20] . fibrosis), Anex6 (impairing regeneration) and genes in the XLMTM is due to X-linked recessive mutations in MTM1 , dyscalc locus (causing calcification) and potentially other encoding myotubularin with important roles in membrane as yet unidentified mutations. In both models, severity trafficking and other intracellular processes. XLMTM is a differs between muscles, with diaphragm being most severely severe and often lethal early-onset myopathy with clinical affected and tibialis anterior least severely affected. Functional features of marked weakness, severe respiratory and bulbar differences (in hanging wire tests) exist between genders, with involvement, as well as the presence of numerous centralized females outperforming males, and BL10-mdx outperforming nuclei and predominance of atrophic type 1 fibres on muscle D2- mdx mice. D2- mdx mice have lower counts of centralized biopsy. nuclei and active regeneration (BrdU and eMHC staining). A These findings, in conjunction with the recent identification striking difference between the strains is extensive calcified of mutations in PAX7 and MYMK (see below), suggest muscles in D2- mdx mice [23] . These seem to be directly that there is a distinct CM subgroup where satellite cell linked to muscle damage, differ in abundance between pathology is crucially involved. These CMs are often muscles and these calcifications ameliorate with age (very characterized clinically by marked axial involvement and abundant in 10 week old mice, but much reduced numbers at severe respiratory impairment, and predominance of atrophic 34 weeks of age). The reason for this is still unknown. type 1 fibres and increases in connective tissue on muscle Lastly she discussed muscle pathology in two mouse biopsy, indicating a consistent phenotype. Assessment of models for LGMD (sgca-null for LGMD2D and sgcd-null for satellite cell involvement (in particular regarding activation LGMD2F). A striking difference between the strains is that states and cell fate commitment) has been variable and muscle function in sgca-null males and females is affected to requires further standardization. The involvement of satellite the same extent, while in sgcd-null mice, females outperform cells in other CMs, and in particular their role in the males [24] . Also, the extent of pathology differs between commonly observed disturbances of muscle growth, remains muscles and ages, although again diaphragm is most severely to be elucidated. affected in these strains. In these models, muscle regeneration is active in the young age (8 weeks), but decreases in adult 2.7. Pathological pathways in dystrophic models of DMD mice (24 weeks). and LGMD: van Putten 3. Models of satellite cell in involvement in muscle van Putten started by pointing out that despite the regeneration many similarities in disease pathology between muscular dystrophy patients and mouse models (i.e., primary defect, The workshop dedicated one session to discuss models chronic cascades of muscle degeneration, regeneration, severe to study satellite cell function in a spectrum of models. In inflammation response and the development of fibrosis), there the mdx mouse, Partridge presented alternative approaches are also many differences. These include amongst others to investigate growth and repair of skeletal muscle in differences in growth speed, distribution of bodyweight on animal models. This consisted of monitoring the rates of limb muscles, a lack of development of fat in mice and a increase in number of myonuclei in fibres extracted from the general disease stabilization in adulthood. Only in animal extensor digitorum longus (EDL) muscle and of measuring models lacking multiple structural muscle proteins, is life the amounts of muscle generated by satellite cells labelled span dramatically reduce. It remains important to keep with BrdU over known time periods. these differences in mind when conducting basic science The former protocol was used to establish the rates of experiments or pre-clinical studies with animal models; they input from myogenic cells over the periods of growth in the remain models each of which have their own limitations. immediate postnatal period, in the juvenile growth period in van Putten then focussed on the muscle pathology in normal wild-type mice and the initial period of muscle repair mdx mice, which is characterized by cycles of degeneration and regeneration in different strains of dystrophic mouse. and regeneration from the age of 3 weeks onwards, after It showed that the contribution of satellite cells to muscle which it stabilizes in adulthood due to their relatively good growth during the 3–4 week postnatal period was closely regenerative capacity. Several attempts have been undertaken comparable in dystrophic mdx and in non-dystrophic wild- J. Morgan, G. Butler-Browne and F. Muntoni et al. / Neuromuscular Disorders 29 (2019) 704–715 709 type mice. It also showed, unexpectedly, that the addition of Notably, the transcription factor Zfh1 is the only identified new myonuclei to normal muscle continued at a slow rate, marker of satellite cells in Drosophila. Interestingly, recent during growth up to 14 weeks of age and that in the mdx work has documented the expression of Zfh1 in mouse mice, myonuclei were added to each fibre at twice this rate satellite cells and evidenced that this gene is required for to achieve a doubling of myonuclei per unit volume. muscle regeneration [26] . Expression of the fly homolog The BrdU labelling investigations showed that, during the of the mammalian satellite cell marker Pax7, has not been early phase of the disease, mdx mouse muscle undergoes a reported in Drosophila. A rigorous molecular comparison and progressive replacement of some 5–10% of its muscle fibres contrast with mammalian satellite cells will become possible every 3 days, and fully regenerates each necrotic lesion on through transcriptomic analyses. the basis of 3 days of myogenic cell proliferation. This To examine changes in morphology that may result from measure of myogenesis demonstrates a clear distinction physical damage or dystrophy, Chaturvedi has standardised a between the very rapid and complete repair of muscle lesions protocol to visualise adult Drosophila muscles in situ in 3- in the C57Bl-mdx mouse from a barely detectable repair dimension, through MicroCT scanning. Quantitation of these process in the DBA/2J-mdx mouse. The latter dystrophic images have revealed definite and consistent differences in mouse model also manifests a severe widely distributed muscle morphology within, and between, male and female calcification of areas of necrosis that are eventually resolved, Drosophila with age. accompanied by an early onset of endomysial fibrosis. These At this workshop, many aspects of human myopathies, features make the DBA-mdx mouse an interesting new animal specifically mutations in dystrophy patients were highlighted. model in which to analyse inhibition of myogenic repair and Further, aspects of Drosophila muscle anatomy that need accompanied by calcification and fibrosis. further exploration for an accurate comparison with human muscular dystrophies became clear. Taken together, the 3.1. Cell transplant models: Morgan identification of adult satellite cells in Drosophila, the new technical ability to accurately measure morphology with the Morgan reviewed pre-clinical work on freshly-isolated plethora with in vivo genetic perturbation techniques, have satellite cells derived from either normal, mdx or all positioned Drosophila as a good in vivo model system for

Large myd mice (models of DMD and dystroglycanopathy human myopathy and muscle repair. respectively). When transplanted into pre-irradiated muscles of immunodeficient mdx mice, freshly isolated donor satellite 3.3. Zebrafish models: Wood cells behave as stem cells, contributing to muscle regeneration and functionally reconstiting the satellite cell pool. Although Wood presented work on three aspects of neuromuscular satellite cell numbers decline over the lifespan and the vast disease research using zebrafish as an animal model. Firstly he majority are activated in growing, 2-week old muscles [25] , discussed that the dystrophin-associated glycoprotein complex donor satellite cells derived from mice from 2 weeks to 2 (DGC) is highly conserved in zebrafish. The small tropical years of age engraft equally well when transplanted into fresh water zebrafish has been used in muscular dystrophy young immunodeficient mdx irradiated muscles. The host research since 2003 when the DMD fish sapje was first muscle environment has a profound effect on donor satellite characterised. Since this time, the most common forms of cell-mediated muscle regeneration, with irradiation being the muscular dystrophy have been identified from ENU mutagenic only host muscle pre-treatment that mediated significant screens or modelled with CRISPRs, TALENs and ZFNs, the engraftment [25] . The environment in which satellite cells field has moved away from antisense morpholino technology reside has a profound effect on their performance, but when despite several well controlled studies [27] . A point of interest removed from the pathological environment, satellite cells was that fish do not have central nucleation on pathology from Large myd mice function well in vitro [4] and satellite with the exception of fish models of laminopathies. Briefly cells derived from aged mdx mice contribute as well to discussed was the fact that FSHD is a particularly problematic functional satellite cells as do satellite cells derived from muscular dystrophy to model in fish. young adult normal mice [3] . Secondly he highlighted that transgenic muscle stem cell and progenitor lines exist. The Tol2 system has been 3.2. Dropophila models: Chaturvedi used to create Tg(pax3a:EGFP), TgBAC(pax7a:EGFP), Tg(met:mCherry-2A-KALTA4), and Tg(myf5:EGFP) Drosophila have recently become a viable model for myf5 lines. Zebrabow line Tg(ubb:LOX2272-LOXP-RFP- studying muscle repair with the demonstration of satellite LOX2272-CFP-LOXP-YFP) crossed onto a tamoxifen cells in adult muscles. Their mononucleate morphology, inducible mesogenin cre Tg(mgsn1: CreERT2) line can be adjacency to muscle fibres, proliferation upon physical used to show in the fish model normal cell division in trauma, fusion of their progeny with damaged muscles, and muscle is asymmetric but in regenerating muscle there is lineage shared with adult mature muscle, motivate further symmetric division at the injury site [28] , in confirmation of inquiry into their function. Specifically, their role in muscle the work on Prof. Terry Partridge’s mouse model presented repair remains to be assessed through (i) satellite cell specific earlier in the session. Currently not many DGC lines have ablation and (ii) satellite cell transplantation experiments. been crossed onto these transgenic lines, despite general 710 J. Morgan, G. Butler-Browne and F. Muntoni et al. / Neuromuscular Disorders 29 (2019) 704–715 enthusiasm, due to the time consuming nature of these maintenance of satellite cell stemness [5] . In unpublished experiments, that would require additional grant funding. work with myoblast and satellite cell cultures, Bonaldo’s team This study would establish overall stem cell contribution found that treatment with soluble COL6 is able to counteract to muscular dystrophy pathology and highlight novelties myogenic differentiation and increase the incidence of Pax7- between muscular dystrophy models. positive cells differentiation, pointing at a biochemical effect Lastly he had a case for fish being a good model to on signalling pathways. Altogether, these data indicate that study fibre detachment in muscular dystrophies. Due to COL6 has a dual function for skeletal muscle –on one hand optical transparency and ex-utero development it is possible it is critical for the regulation of the mechanical properties of to study basement development in various muscular dystrophy muscle, and on the other hand it transduces specific signals fish models. CMD models are particularly well suited to within satellite cells. the zebrafish system. This may be critical in understanding Separate work revealed that COL6 regulates macrophage basement membrane failure in fibre detachment and fibrosis. recruitment and polarization. In unpublished studies, Bonaldo It was noted that Evans blue dye is particularly effective reported that COL6 ablation impairs macrophage recruitment to investigate sarcolemma integrity. Physiology measurements and M2 polarization after muscle injury, pointing at a further such as passive and active force are achievable in fish independent role of this matrix protein in muscle regeneration. models [29] . A question arose around whether it is possible These findings strengthen the concept that COL6 to exacerbate the phenotype. Four options were identified: plays multiple functions for skeletal muscles, and besides cellulose, electroshock and caffeine in larvae; in adult a influencing myofiber homeostasis and regulating satellite cell swim tunnel can be employed. Zebrabox can measure key activities it also plays a critical role in the inflammatory functional swimming parameters. A final point was made process and tissue repair during muscle regeneration. A around understanding the role tenocytes play in fibre stability major point arising during the discussion of this presentation and cross talk with muscle progenitor cells and how little was indeed related to the scant consideration thus far we know about this cell type’s contribution to muscular given to macrophages in muscle regenerative processes dystrophy. both in physiological and pathological conditions, and the need to increase our knowledge about the involvement of 4. Extracellular matrix and fibroblast influence on inflammatory cells in the pathology of muscular dystrophies. satellite cell activity 4.2. Laminins: Ruegg 4.1. Collagen VI as a key extracellular regulator of muscle homeostasis and regeneration: Bonaldo LAMA2- related Muscular Dystrophy ( LAMA2 muscular dystrophy) or merosin-deficient Congenital Muscular Collagen VI (COL6) is an extracellular matrix protein with Dystrophy 1A (MDC1A) is a severe, early onset and a unique structural organization and playing a remarkably fatal muscular dystrophy that is caused by mutations in the broad range of functions in several tissues, including skeletal gene coding for laminin- α2. Muscles of LAMA2 muscular muscle. Although COL6 expression in muscles is mainly dystrophy patients undergo severe muscle degeneration and provided by interstitial fibroblasts, its proper deposition is show a high degree of fibrosis. Laminins are a family critical for myofiber homeostasis, and mutations of COL6 of heterotrimeric molecules that are major components of genes are causative for human congenital myopathies such the extracellular matrix. Laminin- α2 is mainly found in as Bethlem myopathy and Ullrich congenital muscular the basement membrane of the skeletal muscle and in the dystrophy. In the last two decades, a number of studies endoneurium of the peripheral nerve. The lack of laminin- α2 in COL6 null ( Col6a1 –/–) mice have shed light into the results in the compensatory expression of the developmental pathophysiological mechanisms of COL6-related disorders. laminin- α4. Biochemical studies have shown that laminin- COL6 deficiency affects key intracellular pathways, leading α4 lacks the binding to the laminin- α2 receptors, such as to defective regulation of autophagy, accumulation of α-dystroglycan and α7 integrin. In addition, laminin- α4, dysfunctional organelles and spontaneous apoptosis [30 , 31] . in contrast to laminin- α2, does not self-polymerize. Two Based on the close contact of COL6 microfibrillar network specifically designed linker proteins can alleviate both of with the myofiber sarcolemma and its distinctive deposition these shortcomings of laminin- α4. One linker, termed mini- in muscle endomysium, further studies revealed that this agrin, binds to the coiled-coil region of laminin and to matrix molecule plays a pivotal role for satellite cells. α-dystroglycan. The second linker, a chimera between the Indeed, COL6 is a component of the satellite cell niche, LN domain of laminin- α1 and the laminin-binding domain which is expressed by quiescent satellite cells in a regulated of nidogen, allows self-polymerization. When expressed in manner and is required for the proper self-renewal of satellite skeletal muscle fibres, both linkers are incorporated into the cells both in physiological conditions and during muscle muscle basement membrane and confer stable association of regeneration. In vivo and in vitro studies in Col6a1 –/– mice laminin- α4 with the basement membrane and the binding to demonstrated that COL6 is involved in the fine regulation the plasma membrane of the muscle fibres. As a consequence, of the biomechanical properties of skeletal muscle, and the two linkers improve the health of LAMA2 mice to a this is one mechanism through which COL6 influences the large degree as shown in recent proof-of-principle studies J. Morgan, G. Butler-Browne and F. Muntoni et al. / Neuromuscular Disorders 29 (2019) 704–715 711 in mice [32 , 33] . Another feature of the muscle of LAMA2 cells are driven out of the proliferating state and differentiate muscular dystrophy patients is a strong impairment in muscle [37] . Thus, oscillatory MyoD expression allows for the regeneration despite the high extent of muscle degeneration. amplification of the activated stem cell pool to ensure correct This raises the question whether this regeneration deficit is muscle growth and regeneration. Birchmeier investigated a based on cell-intrinsic alterations of muscle satellite cells to mechanism that causes muscular dystrophy when disturbed regenerate or on changes in the environment. Interestingly, the in humans (i.e., aberrant Notch signalling), and demonstrates two linkers although not expressed in muscle satellite cells, the importance of the balanced proliferation/differentiation of greatly improve cardiotoxin-induced muscle regeneration in muscle stem cells in muscle growth and repair. LAMA2 muscular dystrophy, suggesting extracellular cues being important to improve regeneration. In summary, the 5.2. Postnatal regulation of satellite cells: Relaix presented experiments are strong evidence that LAMA2 muscular dystropy is based on the failure to properly In recent years, it has been established that quiescence is assemble muscle basement membrane and to connect it not a dormant state –as previously thought - but rather a to the muscle fibre membrane. The fact that the cDNAs dynamic one, that is maintained by the activity of several encoding the two linkers are small enough to be incorporated different pathways. Stem cells in most adult tissues are into AAV vectors, make it possible to translate this strategy sustained quiescent under homeostatic conditions by the into the clinic. combination of factors that are provided by the specialised microenvironment, also known as the niche. Identifying the 5. Regulation of satellite cells in development, postnatally diverse factors of the niche and their modes of interaction is and in neuromuscular diseases a major challenge and a prerequisite for the use of muscle stem cells in regenerative medicine. One major obstacle 5.1. Oscillations of MyoD and Hes1 proteins regulate the for the study of quiescent cells is that any perturbation of maintenance of activated muscle stem cells: Birchmeier the niche triggers a fast activation, which is manifested by swift transcriptional and epigenetic changes. To overcome The balance between proliferation and differentiation this, Relaix has developed a novel protocol that permits the of muscle stem cells is tightly controlled, ensuring the isolation of quiescent satellite cells in their in vivo state. maintenance of a cellular pool needed for muscle growth and Based on this new approach, they have performed a series repair. Notch signalling provides important cues that regulate of high throughput and single cells experiments that identify this balance. In mice, mutation of components of the Notch the major players of the quiescence and activation networks, signalling pathway result in deficits in muscle growth, a loss including in preclinical models of neuromuscular disorders. of the muscle stem cell pool and severe regeneration deficits Several new approaches to the treatment of DMD are on [34–36] . In humans, mutation of the POGLUT1 (protein the pathway to approval and development. A critical step is O-glucosyltransferase 1) gene which encodes an enzyme first determining efficacy or safety by using an animal model involved in posttranslational Notch modification, causes of the disease. These are most useful in preclinical study an autosomal recessive limb-girdle muscular dystrophy. when they recapitulate the clinical condition as observed in Birchmeier showed during her presentation that the direct human patients. Indeed, dissimilarities between the disease in Notch target gene Hes1 controls the balance between humans and animals may be one cause of the high failure rate, proliferation and differentiation of activated muscle stem cells where therapies appear to improve a condition in the model, in both developing and regenerating muscle of mice. She but show poor clinical efficacy. Relaix therefore generated a showed that Hes1 is expressed in an oscillatory manner in rat model (R-DMDdel52) where the deletion mimics those activated stem cells where it drives the oscillatory expression found in a significant portion of patients, followed by a of MyoD. MyoD expression oscillates in activated muscle rigorous characterisation of the phenotype. Overall, Relaix stem cells from postnatal and adult muscle under various observed a severe and progressive disease, in keeping with conditions: when the stem cells are dispersed in culture, when what is seen in patients affected by DMD, and death around they remain associated with single muscle fibres, or when they one year of age associated with loss of muscle mass. reside in muscle biopsies. When myogenic cells differentiate, R-DMDdel52 rats display dystrophin-deficiency, associated MyoD oscillations become unstable and instead long periods with histopathological changes seen in severely affected of sustained MyoD expression are observed. Similarly, DMD muscle, including increased fibre size variability with when stable MyoD oscillations are experimentally disturbed, the presence of many very small and hypertrophic fibres, for instance by ablation of the Hes1 oscillator, activated increased centronucleation, increased fibrosis and necrosis. muscle stem cells differentiated with higher probability. This Altogether, these results suggest that R-DMDdel52 is a an precocious differentiation interfered with the maintenance of interesting preclinical model for DMD. the muscle stem cell pool, and impaired muscle growth and repair. Analysis indicates that the oscillatory expression of 5.3. Carey-Fineman-Ziter Syndrome: Bonneman MyoD allows activated myogenic stem cells to remain in a proliferative state. However, when MyoD oscillations become This syndrome was originally described in two siblings unstable and are replaced by sustained MyoD expression, with bilateral facial weakness and Robin sequence 712 J. Morgan, G. 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(mandibular hypoplasia, hypoglossia, cleft palate), as well as senescence entry [39] . Since damaged mitochondria are one mild proximal weakness with small muscles, delayed motor of the main sources of reactive oxygen species (ROS), milestones, scoliosis, and normal intelligence. As part of the decrease in mitophagy activity associated with age an investigation into genetic causes for Moebius syndrome led to an increase in oxidative stress. Treatment with biallelic mutations in the gene myomaker/ TMEM8C were antioxidants led to autophagy/mitophagy recovery in vivo , identified in the original family as well as in four additional which in turn prevented the intracellular accumulation of families with a total of 8 patients [6] . The clinical phenotype damaged components and entry into senescence, ultimately was fairly consistent with prominent congenital bifacial restoring the regenerative capacity of aged satellite cells. Of weakness but without prominent ophthalmoplegia, prominent note, Muñoz-Cánoves found that a caloric restriction regime axial weakness and milder involvement of extremity muscles. in old mice increased satellite cell regenerative functions, Muscle biopsy analysis was consistent with fibre type at least in part by promoting autophagy (unpublished disproportion with smal type I fibres and unusually large results). In conclusion, they have shown that autophagy type II fibres. Myomaker has been shown to be critical for is essential to maintain "stemness". Their more recent myoblast fusion, while germline inactivation in the mouse results indicate that autophagic activity is also impeded causes prenatal lethality with muscles consisting of unfused in satellite cells of mdx dystrophic mice, correlating with single nucleated myogenic cells. Notably, none of the human increased cellular senescence (unpublished results). Therefore, genotypes leads to a predicted complete loss of myomaker, enhancing the regenerative capacity of satellite cells through consistent with the notion that a complete loss of myomaker the pharmacological or dietary activation of autophagy, or function would also not be compatible with postnatal human reduction of senescence, opens the possibility to future life. Myomaker is required on both cells involved in the fusion regenerative therapies in the context of muscle regeneration process, while the more recently discovered pore forming problems associated with aging or muscular dystrophies. peptide myomixer/myomerger/minion is only required on one of the two fusing cells. While the knock out mouse 5.5. Novel mouse models to study dystrophin in control and model is histologically very similar to the myomaker knock disease context: Helge Amthor out situation, no human equivalent condition has yet been described. Given that myomaker would also be predicted Dmd mdx is a frequently used mouse model for DMD to be required for satellite cell fusion into the myofiber, and dystrophin-restoring therapies. Amthor made a strong Carey-Fineman-Ziter Syndrome can be conceptualized as a case that current translational research is methologically disorder of muscle satellite cells, with presumed pathogenic limited by antibody-mediated signal amplification to visualize mechanisms in both development as well as in later skeletal dystrophin. Amthor shared unpublished data on Dmd EGFP musclemaintenance. Hypomorphic mouse models will be carrying a C-terminal EGFP-tag and a Dmd EGFP-mdx reporter needed to investigate these important questions in detail. The mouse line carrying additionally in cis the mdx- 23 mutation, selective involvement early on of facial and axial muscles which were generated in collaboration with the laboratory is striking and raises additional interesting developmental of Markus Schuelke in Berlin. These novel models facilitate questions. the study of dystrophin expression/restoration not only at sarcolemmal level, but also in neuromuscular junction, 5.4. Understanding satellite cell regenerative decline with myotendinous junction and muscle stem cells, which are aging: Muñoz-Cánoves central components of skeletal muscle and required for its excitation, force transmission and regeneration. Helge The regenerative function of satellite cells declines with Amthor argued that dystrophin restoration at critical sites of aging. Results from Muñoz-Cánoves’s group have shown expression is required for therapies to be effective. However, that quiescent satellite cells have a constitutive autophagic current standards of dystrophin quantification in preclinical activity that allows them to maintain the correct intracellular models and those from clinical research studies may only be balance between synthesis and degradation of proteins partially addressing this issue. Novel methods are required and organelles [38] . This basal autophagy is essential to to improve imaging of dystrophin restoration not only at the preserve satellite cell homeostasis and proliferative capacity sarcolemma but also other key sites listed to better predict to generate new muscle fibres upon tissue injury. Muñoz- benefits of a therapy. Cánoves observed that autophagy is decreased in satellite cells of aged mice, affecting their internal cleansing process. 5.6. Single cell atlas of aged and dystrophic muscle: As a result, they observed that the intracellular accumulation Giordani of damaged proteins and organelles such as mitochondria could not be eliminated correctly, triggering a switch from Lorenzo Giordani attended the workshop as a special quiescence to a senescence-like state. Muñoz-Cánoves found young scientist delegate. He highlighted that skeletal muscle that pharmacological or genetic restoration of autophagy in repair requires the activation of resident stem/progenitor aged satellite cells allowed them to recover their regenerative cells (satellite cells) that can both self-renew and generate capacity and escape senescence. Conversely, genetic inhibition differentiated progeny. To correctly execute the regenerative of autophagy in quiescent young satellite cells caused program, satellite cells interact with their microenvironment J. Morgan, G. Butler-Browne and F. Muntoni et al. / Neuromuscular Disorders 29 (2019) 704–715 713 and require signals from neighbouring cells. However, in working for the coming three years: Fast Track to treatments, pathologic contexts such as degenerative myopathies (e.g., mental health matters, advances in technology, sport, leisure DMD) the efficiency of this process progressively declines and exercise. leading ultimately to incomplete repair, fat infiltration and The charity also funds research into the causes and fibrotic scarring. Understanding the cellular mechanisms potential treatments for these conditions, from basic to behind regeneration impairment is pivotal to the development preclinical to clinical studies. Funding for research projects of novel strategies aimed at slowing down and counteract is primarily for UK researchers and is awarded through disease progression. Standard bulk-scale methods had been a rigorous, competitive process involving peer review and proven unfit to address these questions since are unable assessment by the charity’s Medical Research Committee to correctly render the heterogeneity within the populations and Lay Research Panel. Lay summaries must be provided involved in tissue repair. As a result, defective disease-specific by applicants and are scored by the Lay Research Panel subpopulations remain most of the time undetected. Hence members, making this a crucial part of the selection process. the need to apply a single-cell approach to identify those Alison finished by reflecting on the questions raised by dysfunctional subsets. members of the Lay Research Panel around stem cells in Giordani presented a two-step strategy to study the muscular dystrophy. Their comments focussed on the need different resident populations in skeletal muscles and identify and desire for a potential treatment, and questions were raised those potentially involved in the regeneration impairment. about how this would work. For example, what would a stem First single-cell transcriptomics and mass cytometry (CyTOF) cell treatment look like, what would the source of stem cells analyses have been used to attain a clearer understanding be, how often would a treatment be administered and how of the cellular composition of adult skeletal muscle, leading would a balance be reached between the need for immune to the identification of muscle resident tenocyte-like cells suppression and the risk of infection? Other considerations (SCX + ) as well as a non-multipotent myogenic subset of were: who would be eligible (treatments should be inclusive mesenchymal helper cells. Then, using different algorithms of children) and how to ensure a quick route to market. Alison (SPADE, Statistical Scaffold) Giordani overlaid the initial acknowledged that much research is currently at a basic level, single cell blueprint on both the dystrophic (MDX 4cv) and rather than developing stem cells as potential treatments, but the aged condition. This revealed functional similarities and noted it would be important to bear these comments and highlight recurring patterns. Lastly, as proof of concept he questions in mind for the workshop discussions and for future showed some preliminary data on CyTOF on human biopsies. research. The data presented demonstrate that this approach could be successfully applied to any tissue. In particular, in pathologic 6.2. Patient’s perspective: Davenport conditions, it could serve to precisely identify dysfunctional subfractions [40] . Richard Davenport explored some personal reflections on This two-step approach could be used to study the specific the effects of muscular dystrophy on his life, pain, rehab role of satellite cells and other skeletal muscle population in and the many changes it brings. He suggested that self- the pathology of muscular dystrophy. Presenting single cell image, our perceptions of ourselves and how we fit into the technologies (such as mass cytometry) to clinicians could be world, can be really influential in managing the condition but instrumental to build a collaborative network to successfully also challenged by those changes. So much so, that bigger apply this approach to different specific muscular dystrophies. physical changes leave our self-image with a gap in meaning and a certain amount of reinvention and resocialisation takes 6. Funder’s and patient perspective place to fill it. Because life with muscular dystrophy is relentless change and adaption, regular focused counselling, 6.1. Funder’s perspective: Stevenson from diagnosis, could greatly assist patients in proactively managing these sometimes existential challenges. Alison Stevenson, Senior Grants Manager at Muscular Aside from the individual-focused changes and challenges, Dystrophy UK, spoke about the charity’s work for people with Richard also talked about how access becomes critical but the neuromuscular conditions, its research strategy and funding prevalent attitude to access is one of ineffective apology or opportunities. flat nonchalance. These sorts of "social model" issues become Muscular Dystrophy UK is the UK’s largest charity for a daily grind as much as the physical struggles. people with muscular dystrophy and related neuromuscular conditions, and has an annual income of over £7million. 7. Final remarks Approximately 70,000 people in the UK are affected by muscular dystrophy or a related neuromuscular condition. This workshop has shed light on stem cell involvement The charity provides information, advocacy and other in the pathogenesis of muscular dystrophies and congenital support to enable individuals affected by these conditions to myopathies, but we need to gain a greater understanding of: live as independently as possible, because for people living with a muscle-wasting condition every day counts. Muscular • The involvement of satellite cells in experimental models Dystrophy UK’s recent strategy outlines four priority areas of and humans, particularly the importance of rigorously 714 J. Morgan, G. Butler-Browne and F. Muntoni et al. / Neuromuscular Disorders 29 (2019) 704–715

evaluating all in vitro data in the context of what actually Maaike Van Putten (Leiden, The Netherlands), Alasdair Wood occurs in vivo. (Monash, Australia), Peter Zammit (London, UK). • The fate and biology of satellite cells during ageing and in disease. For old muscles, a large body of data Acknowledgements indicate no major intrinsic problem in myogenic capacity of satellite cells, especially in vivo : this is controversial. This Workshop was made possible thanks to the financial However, major age-related alterations to the inflammatory support of the European Neuromuscular Centre (ENMC) and cell response are well documented, with adverse effects ENMC main sponsors: on the kinetics of regeneration after experimental injury - Association Française contre les Myopathies (France) of old muscles. Furthermore, there appears to be minimal - Deutsche Gesellschaft für Muskelkranke (Germany) incidence of necrosis/ regeneration in healthy old human - Muscular Dystrophy Campaign (UK) and mouse muscles (and thus no ongoing myogenesis and - Muskelsvindfonden (Denmark) regenerating host myofibres to fuse with donor myoblasts - Prinses Beatrix Spierfonds (The Netherlands) in any proposed stem cell therapy). - Schweizerische Stiftung für die Erforschung der • The role of satellite cells in development of muscle Muskelkrankheiten (Switzerland) pathology in DMD, Limb Girdle Muscular Dystrophy and - Telethon Foundation (Italy) other muscular dystrophies. Much data indicate no major - Spierziekten Nederland (The Netherlands) and Associated intrinsic myogenic stem (satellite) cell problem in vivo members: for DMD and LGMD2B. The controversy over stem cell - Finnish Neuromuscular Association (Finland deficits in DMD may be due to differences in vivo and in With a special thanks to the Muscular Dystrophy UK for vitro environments. their support • The impact of metabolism on the muscle repair This work is (partly) funded by the NIHR GOSH BRC programme. The work presented here showed that reducing (FM and JEM). The views expressed are those of the author(s) organismal energy levels promotes satellite cell function in and not necessarily those of the NHS, the NIHR or the mouse models of DMD, whereas other previous work has Department of Health. documented the benefits of high fat diets in supporting muscle fibre repair. Supplementary materials • The evidence of defective satellite cells as primary cause of EMARDD and CFZ Syndrome. Supplementary material associated with this article can be • The efficacy of therapeutic strategies to correct satellite found, in the online version, at doi: 10.1016/j.nmd.2019.07. cell dysfunction in muscular dystrophies. For this to be 003 . effective, any intrinsic problem with satellite cells in dystrophic muscles in vivo must be fully understood. Major References problems are well-documented for stem cell transplantation

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